Systemic fungal infections are a major cause of mortality in cancer patients and other immunocompromised individuals. Unfortunately, fungal infections very often defy treatment because the few drugs that destroy fungi are extremely toxic to the host. Because of the drugs' toxicity, the lowest possible effective doses should be given. Unfortunately, because the drugs are diluted in the blood, and because large amounts of the drugs are degraded, or excreted or taken up by uninfected tissue, large doses actually are and must be given if the treatment is to be effective.
The preferred treatment for systemic fungal infections is primarily limited to two groups of drugs: the polyene antibiotics such as Amphotericin B and nystatin, and the imidazoles, such as ketaconazole and miconazole. The polyene antifungal antibiotics readily bind to sterol components of host cells causing disruption of the membrane, cell permeability and lysis. Amphotericin B has thus been associated with acute hemolytic crisis. Further, because it is particularly toxic to kidney tissue, it has been associated with irreversible renal damage and even kidney failure, at therapeutic dosage levels. Medoff, G., Kabayashi, G. (1980) Strategies in treatment of systemic fungal infections. New England Journal of Medicine 302: 145-55; Cohen, J. (1982) Antifungal chemotherapy. Lancet ii: 532-37; Graybill, J. R., Craven, P. C. (1983) Antifungal agents used in systemic mycosis: activity and therapeutic use. Drugs 25: 41-62.
It is a major goal of medical research to overcome the problems presented by the need for compromise between dosages high enough to control infection on the one hand, and unacceptable damage to healthy tissues on the other. Recently it has been discovered that needed doses of medicine can be delivered to diseased tissue while bypassing healthy tissue using certain liposomal formulations. Additionally, it has been recognized that medication can be incorporated into liposomes, microscopic delivery vesicles made, in part, of phospholipids. See U.S. Pat. No. 4,663,167--Composition and Method for Treatment of Disseminated Fungal Infections in Mammals, incorporated here by reference, and pending Vestar Research Inc. application Ser. No. 899,064, entitled "Improved Treatment of Systemic Fungal Infections With Phospholipid Particles Encapsulating Polyene Antifungal Antibiotics", also incorporated here by reference, which discloses liposomal delivery vesicles made, in part, from phospholipids.
Phospholipids form closed, fluid filled spheres when mixed with water. Phospholipid molecules are polar, having a hydrophilic ionizable head, and a hydrophobic tail consisting of long fatty acid chains. Thus, when sufficient phospholipid molecules are present with water, the tails spontaneously herd together to exclude the water while the hydrophilic phosphate heads form bonds with the water.
The result is a bilayer in which the fatty acid tails point into the newly formed membrane's interior and the polar heads point toward the aqueous medium. The polar heads at one surface of the membrane point toward the liposome's aqueous interior and those at the other surface point toward the aqueous exterior environment. It is this chemical tendency to form liquid filled spheres that allows the liposome to be loaded with medication. As the liposomes form, water soluble molecules will be incorporated into the aqueous interior, and lipophilic molecules will tend to be incorporated into the lipid bilayer. Liposomes may be either multilamellar, like an onion with liquid separating many lipid bilayers, or unilamellar, with a single bilayer surrounding an entirely liquid center.
In studies of mice, Amphotericin B incorporated into liposomes has been shown to treat systemic fungal infections more effectively than when given as the free drug. Liposomes are not themselves toxic, and they protect their loads from being degraded or diluted. Thus, liposomes are thought to deliver concentrated doses of antifungal antibiotic at the diseased tissue without the toxicity that would otherwise be associated with freely circulating drug. Therefore, liposomal Amphotericin B drug doses can exceed the maximum tolerated dose of free Amphotericin B. Mehta, R. (1982). Liposomal Amphotericin B is toxic to fungal cells but not to mammalian cells. Biochimica et Biophysica Acta 770: 230-34. Liposomal encapsulated Amphotericin B has also been shown to be an effective treatment for murine systemic fungal infections, including Candidiasis, Cryptococcosis, and Histoplasmosis. Graybill, J. R. et al (1983) Treatment of murine cryptococcosis with liposomal associated Amphotericin B. Journal of Infectious Diseases 145: 748-52; Taylor, R. L. et al (1982) Amphotericin B in liposomes: A novel therapy for histoplasmosis. American Review of Respiratory Diseases 125: 610-611.
Liposomal Amphotericin B has also shown effectiveness in human patients, life saving when other treatments have failed, including freely circulating Amphotericin B. Systemic fungal infections are seen most commonly in people whose immune systems are compromised by disease or immunosuppressive drug therapy. As previously mentioned, these infections are a common cause of death to victims of acquired immune deficiency syndrome and to cancer patients undergoing chemotherapy. The causative agents of these fungal infections are often endogenous fungi that would be rendered harmless but for the patient's impaired resistance. Lopez-Berestein, G. et al (1987) Treatment of hepatosplenic Candidiasis with liposomal Amphotericin B. Journal of Clinical Oncology 5: 310-17.
Unfortunately, because of the chemical properties of the polyene antifungal antibiotics, it has heretofore not been possible to produce liposomal Amphotericin B in commercial quantities. These antifungal agents are called polyenes because they contain three to seven conjugated double bonds in the aliphatic chain making up a large lactone ring. The double bonds are incorporated into one side of the ring of 26 to 44 carbon atoms and along the opposite side of the ring 6 to 12 hydroxyl groups are present. Additionally, these molecules contain specific carboxylic acid groups and amine groups. Amphotericin B and nystatin, for example, possess both an aminosugar and a carboxylic acid group. The polyene regions of the molecules are of course hydrophobic and lipophilic while the polyol and ionizable regions are hydrophilic and lipophobic. As such, these molecules are called amphiphilic. Additionally, because of the carboxylic group and the amine group, Amphotericin B can act as a Lowry-Br.o slashed.nsted acid or proton donor, or as a Lowry-Br.o slashed.nsted base or proton acceptor. The combination of these functionalities makes polyenes very poorly soluble in water and most organic solvents. Bennett, J. E. (1974) Chemotherapy of systemic mycoses. New England J. Medicine 290: 320-23.
It has been the persistent problem of insolubility of the polyene antifungal antibiotics in general and of Amphotericin B in particular that has previously limited the prior art. Liposomal Amphotericin B formation was heretofore limited to generally two methods, described below, neither feasible for commercial scale production and neither showing long-term stability or as great a reduction in toxicity as the preparations herein described.
One prior method requires that the Amphotericin B be first dissolved in large volumes of volatile organic solvent such as methanol. To that solution would then be added the lipid mixture dissolved in a volatile organic solvent such as methanol and/or chloroform. The solvents would then have to be removed from the mixture to form a lipid-Amphotericin B film. Removal of solvents could be accomplished by a variety of methods but usually by evaporation to dryness in a round bottom flask under vacuum or nitrogen. Taylor, R. L. (1980) Amphotericin B in liposomes: A novel therapy for histoplasmosis. Am. Review Respiratory Disease 125: 610-11; Graybill, J. R. et al (1982) Treatment of murine crytococcosis diseases. J. Infectious Diseases 145: 748-52; Lopez-Berestein, G. (1983) Treatment and prophylaxis in disseminated infection due to Candida albicans in mice with liposome-encapsulated Amphotericin B. J. Infectious Diseases 147: 939-45; U.S. Pat. No. 4,663,167--Composition and method for treatment of disseminated fungal infections in mammals. The prior art methods thus required removal of large volumes of organic solvent, and preparation of a lipid-Amphotericin B film, an extra step eliminated by the present invention. Moreover, the prior art methods were thus practicable only in discrete batches and were not amenable to the continuous flow process of the current invention. These are two disadvantages which the industry has long attempted to overcome.
In another method of forming liposomal Amphotericin B, the lipid mixture is dissolved in chloroform or another solvent and deposited and dried on the sides of a round bottom flask or vesicle surface. A solution of Amphotericin B dissolved in a small amount of dimethyl sulfoxide would then be added to the previously deposited lipid film. The resulting preparation would thereafter have to be dialyzed against a buffered saline or other solution to remove the dimethyl sulfoxide and non-intercalated Amphotericin B. The procedure was extremely time consuming and expensive, and typically resulted in incorporation of only 70% of the initial Amphotericin B. Tremblay, C. et al (1984) Efficacy of liposome-intercalated Amphotericin B in treatment of systemic Candidiasis in mice. Antimicrobial Agents and Chemotherapy 26: 170-73.
Prior to the present invention, it was not possible to dissolve Amphotericin B in small quantities of volatile solvent, such that scaled-up production could be commercially feasible. The present invention also enables the dissolved Amphotericin B--phospholipid liposomal solution to be spray dried, thus making commercial quantities practical by elimination of the elaborate and time consuming steps detailed above.
Accordingly, one object of the present invention is to provide an improved process for the solubilization of amphophilic drugs.
Another object of the present invention is to provide an improved process for the encapsulation of polyene antifungal antibiotics into liposomes.
More specifically, an object of the present invention is to provide an improved process for the formation of liposomal Amphotericin B.
Yet another object of the present invention is to provide a commercially feasible process for the production of liposomal Amphotericin B.
Yet another object of the present invention is to provide a process for the formation of liposomal Amphotericin B with reduced toxicity.
A further object of the present invention is to provide new methods of treatment with Amphotericin B.
The manner in which these and other objects are realized by the present invention will be apparent from the summary and detailed description set forth below.